棉花GhFUL1基因及其启动子的克隆和功能分析

Cloning and Functional Analysis of GhFUL1 Gene and Its Promoter in Cotton

为探究GhFUL1基因在棉花分枝发育进程中的功能,从棉花中克隆MADS-box家族GhFUL1基因及其启动子,对该基因进行功能研究,并对其启动子进行活性分析。结果表明,棉花中GhFUL1基因开放阅读框726 bp,编码241个氨基酸。进化树分析表明,GhFUL1与其他物种中可可的TcAGL8-1亲缘关系最近。烟草亚细胞定位分析结果表明,GhFUL1定位在细胞核和细胞膜。不同组织材料qRT-PCR分析表明,GhFUL1基因在茎尖中表达量最高。构建过表达载体转化拟南芥,结果表明,转基因拟南芥阳性株系中GhFUL1基因表达量显著增加,转基因株系的开花时间、花器官形态与野生型相比无明显变化,但是开花时侧枝数增加。qRT-PCR结果表明,细胞分裂素氧化/脱氢酶基因AtCTK1和AtCTK6表达量减少,推测GhFUL1基因可能通过调控细胞分裂素合成途径影响分枝。利用启动子分析网站PlantCARE预测可知,GhFUL1启动子区域不仅有TATA-box、CAAT-box核心元件,还有参与光响应、生长素响应以及胁迫应答等顺式作用元件。将GhFUL1启动子构建到表达载体pBI121检测其启动子活性,通过对拟南芥转基因阳性株系进行GUS染色,发现GhFUL1启动子在拟南芥幼苗期的茎尖分生组织和成熟期的花器官中特异性表达。初步揭示GhFUL1基因及其启动子转化拟南芥后在其分枝发育过程中具有一定的功能。

To investigate the function of GhFUL1 gene in branching development of cotton,the GhFUL1 gene in the MADS-box family and its promoter were cloned from cotton,and the function and promoter activity of the gene were analyzed.The results indicated that the open reading frame(ORF)of GhFUL1 gene was 726 bp and encoded 241 amino acids.Phylogenetic tree analysis showed that the amino acid sequence of FUL subclade was conserved,and GhFUL1 owned the highest similarity to Cacao TcAGL8-1 except for the homologous proteins in cotton.Subcellular localization result showed that GhFUL1 was localized in nucleus and membrane.The expression level of different cotton tissues was examined,and the result showed that GhFUL1 was highly expressed in shoot apical meristem.Overexpressing GhFUL1 in Arabidopsis thaliana increased the number of lateral branches by decreasing the expression of cytokinin oxidation/dehydrogenase genes AtCTK1 and AtCTK6.Compared with the wild type,transgenic plants did not result in significant changes of the flowering time and floral organ development.The present results suggested that GhFUL1 might affect the number of branches by regulating the cytokinin synthesis pathway.The promoter of GhFUL1 was predicted to include the TATA-box and CAAT-box elements,and cis-acting elements related to light response,auxin response and stress response by PlantCARE website.Then the promoter of GhFUL1 was cloned and constructed into expression vector pBI121 to detect promoter activity.The GUS staining of transgenic Arabidopsis lines showed that GhFUL1 promoter was specifically expressed in shoot apical meristem at seedling stage and flower organ at maturity.This study revealed that the gene and promoter of GhFUL1 played an important role in the Arabidopsis branching development process after transformation.